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Contribution of Individual Amino Acids to the RNA Binding Activity of the Wilms’ Tumor Suppressor Protein WT1† Tristen C. Weiss and Paul J. Romaniuk* Department of Biochemistry and Microbiology, UniVersity of Victoria, P.O. Box 3055, Victoria, British Columbia V8W 3P6, Canada ReceiVed August 22, 2008; ReVised Manuscript ReceiVed NoVember 14, 2008
ABSTRACT:
In addition to binding to DNA, the zinc finger protein WT1 can also bind specifically to RNA. To determine the role of individual zinc fingers of the protein in this RNA binding activity, deletion and substitution mutants of the WT1 zinc finger domain were constructed. The effects of the various mutations on the binding of WT1 to the RNA aptamers RNA22 and RNA38 were determined using a quantitative equilibrium binding assay. The results indicate that zinc fingers 2 and 3 of WT1 are essential for the binding of the protein to the RNA aptamers. For both of these fingers, the arginine residue immediately preceding the R-helix makes a significant contribution to RNA binding. For zinc finger 2, a second arginine residue within the R-helix is also critical for RNA binding, while several R-helical residues in zinc finger 3 contribute to the overall affinity of WT1 for RNA. Investigating the effects of the same point mutations on DNA binding indicates that there are similarities and differences in the contributions of zinc fingers 2 and 3 to the DNA and RNA binding activities of WT1.
Zinc finger proteins carry out diverse functions in the cell, generally involving specific interactions with DNA, RNA, or protein ligands. Some members of this class of proteins are multifunctional, which requires that the zinc finger domains in these proteins interact with more than one class of ligand. For example, the Wilms’ tumor suppressor protein WT1 acts as a transcriptional regulator by binding to specific sites in DNA via a domain with four zinc fingers (1, 2). It is also believed to be involved in downstream transcript processing or regulation by virtue of a putative RNA binding activity located in the zinc finger domain and the presence of the protein in poly(A)+ RNPs (3, 4). Further complexity in the function of the WT1 zinc finger domain has been implied by the observation that this domain interacts with a number of protein partners (5-7). The function of the zinc finger domain of WT1 is controlled to some extent by an alternative splicing event that produces two isoforms of the domain, distinguished by the presence (+KTS) or absence (-KTS) of an additional three amino acids in the spacer between zinc fingers 3 and 4 (8). The -KTS isoform colocalizes in the nucleus with regions of active transcription and is capable of binding to DNA, RNA, and protein (9). The +KTS isoform is capable of binding to RNA and proteins and colocalizes with splicing machinery in nuclear speckles (9). The interaction of the WT1(-KTS) zinc finger domain with consensus DNA sequences has been extensively studied by biochemical approaches (10-17), culminating with the solution of a three-dimensional structure for the complex formed between the WT1 zinc finger domain and a double† This work was supported by a grant from the Natural Sciences and Engineering Research Council of Canada. * Corresponding author. Telephone: (250) 721-7088. Fax: (250) 7218855. E-mail:
[email protected].
stranded DNA (18). The potential interaction of WT1 with RNA, and the biological significance of such an interaction, has not been completely elucidated. WT1 has been reported to interact with an intronic region of the IGF-II mRNA (19) and with the 5′ leader region of the actinin-1 mRNA (20). A more defined system for studying the RNA binding activity of WT1 was developed by Pelletier and colleagues. A series of WT1-specific RNA aptamers were isolated from a random library, and the binding of these aptamers to WT1 was characterized (10). Further biochemical investigations demonstrated that the WT1 zinc finger domain derived from both -KTS and +KTS isoforms bound to several of the aptamers with high affinity and specificity, and the binding to the RNA22 aptamer required specific sequence and structural features of the RNA (21). Whereas the role of each zinc finger in the interaction of WT1 with DNA is well characterized, this remains an unresolved question about the RNA binding activity of WT1. Deletion of either the N-terminal or C-terminal zinc finger greatly diminishes the affinity of the WT1 zinc finger domain for the RNA aptamers and the two putative in ViVo RNA targets, leading to the conclusion that an intact zinc finger domain is required for RNA binding (10, 19, 20). In order to understand this phenomenon further, we have created a series of deletion, finger swap, and point mutations in the zinc finger domain of WT1(-KTS) to ascertain the role of each of the zinc fingers in binding to the WT1-specific RNA aptamers. The effects of these mutations on RNA and DNA binding have been determined quantitatively using an equilibrium binding assay. The results indicate that there is an overlap in the roles of certain amino acids in binding to RNA and DNA.
10.1021/bi801586a CCC: $40.75 2009 American Chemical Society Published on Web 12/17/2008
RNA Binding by Zinc Finger Protein WT1
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MATERIALS AND METHODS Bacterial Strains and Plasmid Vectors. The plasmid vector pET30a was used to express recombinant wild-type and mutant WT1(-KTS) proteins in Escherichia coli strain BL21(DE3) using methods that have been described elsewhere (14, 15, 21). Construction of Mutant WT1(-KTS) Expression Vectors. All mutants used in this study were constructed by the polymerase chain reaction (PCR). Amino- and carboxyterminal truncation mutants of the zinc finger domain of WT1(-KTS) were constructed using upstream primers containing a recognition sequence for the restriction enzyme NcoI and downstream primers containing a recognition sequence for the restriction enzyme EcoRI. PCR products were digested with NcoI and EcoRI and ligated into plasmid pET30a that had been digested with the same restriction enzymes. Ligation reactions were used to transform E. coli strain DH5R, and putative constructs were identified by colony PCR using primers specific to pET30a sequences flanking the multiple cloning site. Plasmids were isolated from appropriate colonies, and the correctness of the plasmid constructs was confirmed by DNA sequencing before protein expression and purification. Substitution mutants of WT1(-KTS) zinc finger peptides were constructed by overlap extension PCR (22). For the finger swap mutants, the region of the Xenopus p43 cDNA encoding zinc fingers 6-9 was used as a donor of zinc finger encoding cassettes, and primers were designed that contained base sequences that encoded the junction between the donor p43 finger and the acceptor WT1 finger. Figure 1A shows a comparison of the sequence of the WT1 zinc fingers compared to the last four zinc fingers of p43, and Figure 1B shows the deletion and finger swap mutations that were constructed. Alanine point mutants were created with primers encoding the desired codon change. Final PCR products were digested with NcoI and EcoRI and then ligated into pET30a as described above. Expression and Purification of Recombinant Wild-Type and Mutant Proteins. Preparation of N-terminal His-tagged wild-type and mutant WT1(-KTS) zinc finger peptides was carried out as described previously (14, 15, 21). Protein purity was confirmed by polyacrylamide gel electrophoresis, and the concentration of each protein preparation was determined by the method of Bradford (23). Radiolabeling of RNA and DNA Ligands. Preparation of radiolabeled WT1 aptamers RNA22 and RNA38 by in Vitro transcription was carried out as described previously (21). A DNA restriction fragment with a copy of the WT1 consensus binding site was 3′ end labeled as described previously (14, 15). The sequence of the WT1 consensus DNA binding site and the predicted secondary structures of RNA22 and RNA38 are shown in Figure 1C. Equilibrium Binding of Nucleic Acids to Mutant WT1(-KTS) Proteins. The apparent association constants for the binding of radiolabeled nucleic acids to wild-type and mutant WT1(-KTS) proteins were determined using a double filter binding assay (24). The binding buffer consisted of 20 mM Tris-HCl, pH 7.5 (20 °C), 5 mM MgCl2, 100 mM KCl, 10 µM ZnCl2, 0.5 mM tris(2-carboxyethyl)phosphine hydrochloride, 100 µg/mL BSA, and 1 µg/mL poly(A). When binding of the proteins to DNA was measured, poly(dI-dC)
FIGURE 1: (A) Comparison of the amino acid sequence of the four zinc fingers of WT1(-KTS) with zinc fingers 6-9 of Xenopus p43 used to create finger swap and scanning mutations in WT1. The boundaries of the R-helical region of each finger are indicated by the arrows, and the amino acid residue numbers from full-length WT1 are shown above the sequence. (B) Schematics of deletion and finger swap mutations created in the WT1(-KTS) zinc finger domain peptide. (C) The consensus DNA binding site for WT1(-KTS) used in this study. The secondary structures of the RNA22 and RNA38 aptamers predicted by mfold (39).
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FIGURE 2: Equilibrium binding of wild-type WT1, the four finger peptide p6-9 derived from Xenopus p43 protein, and the finger swap mutants Wp1, Wp2, Wp3, and Wp4 to RNA22 (panel A) and RNA38 (panel B). Each data point is the mean value from three independent experiments, with the standard deviation for each point represented by the error bars. Best-fit curves to a simple bimolecular equilibrium are shown for each protein.
replaced poly(A) in the binding buffer. Apparent dissociation constants (Kd) for the binding of the mutant and wild-type proteins to RNA were calculated by fitting the data to a simple bimolecular equilibrium model using the general curve fitting function of Kaleidagraph software (Synergy Software, Reading, PA) and the equation: [protein]total [RNA-protein] ) [RNA]total [protein]total + Kd where [RNA]total , Kd and [RNA-protein]/[RNA]total is reported as the fraction of RNA bound. Data for the equilibrium binding of proteins to DNA were analyzed in the same way. Association constant (Ka) values were derived as the reciprocal of the measured Kd values and are reported as the mean of three independent determinations with the associated standard deviations. Relative affinities were arrived at by dividing the apparent Ka for the mutant protein by the apparent Ka for the wild-type protein determined in parallel. Apparent Ka values for the binding of the wild-type protein were determined to be (1.42 ( 0.22) × 108 M-1 for RNA22, (2.05 ( 0.14) × 108 M-1 for RNA38, and (2.15 ( 0.29) × 108 M-1 for consensus DNA. The errors for relative affinities are given by the expression σ ) [(σ1/M1)2 + (σ2/ M2)2]1/2 × M2/M1, where M1 and M2 are the respective association constants for the wild-type and mutant protein and the σ values are the corresponding standard deviations for these determinations. In each case, the same preparation of protein was used to measure the affinities to all three ligands. RESULTS Both isoforms of the WT1 zinc finger domain have been shown to bind to RNA aptamers and fragments derived from two cellular RNAs (10, 19, 20), while only the -KTS isomer binds to a 12 base pair consensus DNA with high affinity (14). The equilibrium binding of WT1(-KTS) to DNA and the RNA aptamers is similarly affected by conditions such as monovalent salt concentration, pH, and temperature (21).
Table 1: Effects of Deletion, Broken Finger, and Finger Swap Mutations on the Binding of WT1 to RNA Aptamers protein
relative Ka for RNA22
relative Ka for RNA38
WT1 WT1∆F1 H343A WT1∆F4 H431A Wp1 Wp2 Wp3 Wp4 p6-9
1.00 0.133 ( 0.023 0.508 ( 0.102
